There are several forms of assay devices presently found in the medical diagnostic field that are used for determining a specific analyte of a bodily fluid sample, such as whole blood, by reacting the fluid sample with at least one reagent and then determining an analyte or marker of interest. For example and referring to FIG. 1, there is shown a known lateral flow assay device 1 defined by a substrate 6, which is substantially planar and further defined by an upper or top surface 7, the substrate forming a support. A plurality of projections 12 extend upwardly from the top surface 7. These projections 12 are disposed in a predetermined spaced relation to one another and dimensioned so as to induce lateral capillary force upon a liquid sample that is introduced into the assay device 1. The assay device 1 is further defined by a plurality of areas or zones that are linearly disposed along at least one fluid flow path. More specifically, the assay device 1 includes a sample addition zone 2 adjacent at least one reagent zone 3, the reagent zone 3 having a detection material (not shown), such as a detection conjugate that is coated, impregnated or otherwise applied or deposited onto the projections 12. A flow channel 4 extends from the reagent zone 3 to an absorbing or wicking zone 5 that is disposed at the opposing end of the fluid flow path relative to the sample addition zone 2. Each of the above noted zones according to this design include a plurality of the projections 12 in order to induce lateral capillary flow through the assay device 1, and more specifically along the defined fluid flow path. Additional specifics relating to this lateral flow assay device can be found in U.S. Pat. No. 8,025,854 B2, WO2003/103835, WO2005/089082, WO2005/118139, and WO2006/137785, all of which are incorporated herein by reference in their entireties.
In terms of overall operation, a fluidic sample such as whole blood is initially applied to the sample addition zone 2 through a cover (not shown) or through direct application using a pipette (not shown) or other dispensing means, wherein sample is caused to move along the defined fluid flow path through the reagent zone 3 based on the capillary pressure exerted by the plurality of projections 12. The sample upon encountering the detection material in the reagent zone 3 which, upon contact, therewith produces a detectable signal, such as a color change that is visually perceivable. The sample, along with the gradually dissolved detection material, continues to migrate through the assay device 1 along the defined fluid flow path through the flow channel 4, the latter having at least one predetermined area or zone configured for detection by an instrument, such as a scanning fluorimeter, and wherein the sample continues to move along the fluid flow path to the absorbing zone 5. After a sufficient time to fill the absorbing zone 5, the assay is considered to be complete and a detectable result can be obtained at the predetermined detection area(s) using the detection instrument.
Another example or version of a lateral flow assay device 20 is illustrated in FIG. 2, the device 20 including a planar substrate 40 which can be made from a moldable plastic or other suitable non-porous material. The substrate 40 is defined by a top or upper surface 44, which is further defined by a plurality of discrete zones or areas including a sample receiving zone 48, a reagent zone 52, and an absorbing or wicking zone 60. According to this known device design, each of the above-noted zones are fluidically connected to one another in a linear fashion along a defined fluid flow path that further includes a flow channel 64, which can include at least one detection zone (not shown) and in which a plurality of projections (not shown), similar to those provided in the assay device 1 of FIG. 1, are disposed within at least one of the zones and/or the flow channel 64, the projections extending upwardly from the upper surface 44 of the substrate 40 and in which the projections may be provided in at least one or all of the disposed zones of the assay device 20 to promote sample flow.
The projections can be sufficiently dimensioned so as to spontaneously induce capillary flow without the need for additional structure (i.e., side walls, cover or lid) or the application of any externally applied forces. According to this design, a defined fluid flow path is created from the sample receiving zone 48 extending to the wicking zone 60 and in which the fluid flow path is at least partially open. In another embodiment, the assay device 20 can be entirely open. By “open” what is meant is that there is no cover or lid which is maintained at a distance that would contribute to capillary flow. Thus a lid, if present as physical protection for the flow path and the device 20, does not contribute to the capillary flow produced along the fluid flow path. In this known assay device 20, a hydrophilic foil layer 70 is adhesively or otherwise applied to the top of the projections in the wicking zone 60 in order to increase fluid flow in the assay device 20 and in which a plurality of vents 72 are further defined in the hydrophilic foil layer 70. A flow bridging structure 57 may be provided to further enable flow across an outer edge of the hydrophilic foil layer 70, as further discussed herein. An open lateral flow path is described including the defined projections in the following published application: WO2003/103835; WO2005/089082; WO2005/118139; WO2006/137785; and WO2007/149042 as well as U.S. Patent Application Publication No. 2014/0141527 A1, each of which are herein incorporated by reference in their entireties. More specifically, the extending projections each have a height (H), diameter (D) and a distance or distances between the projections (t1, t2) such that lateral capillary flow of an applied fluid, such as plasma, preferably human plasma, can be achieved. These relationships are further discussed in US Patent Application Publication No. 2006/0285996, which is further incorporated herein by reference in its entirety.
In use, the assay device 20 operates similarly to the assay device 1, FIG. 1, in which a sample is applied to the sample receiving zone 48, which causes sample to move under capillary force to the reagent zone 52 containing the deposited detection material. When wetted by the sample, the detection material may react, depending on the type of assay (e.g., competitive, sandwich, etc.) with the sample and dissolves, thereby producing a visually perceivable (colored) signal. The sample and the dissolved detection material advance along the defined fluid flow path along the flow channel 64 via the projections and under capillary force into the absorbing zone 60. When the absorbing zone 60 is filled with fluid, the assay is assumed to be completed and the assay results can be taken by a detection instrument (e.g., a fluorimeter) relative to the flow channel 64 and at least one detection zone 56.
Referring to FIG. 3, there is depicted yet another known lateral flow assay device 100 defined by a planar substrate 104 which can be made from a moldable plastic or other suitable non-porous material. A plurality of discrete zones or areas are defined in spaced relation along on a top surface of the substrate 104, the zones extending along a linear fluid flow path. These zones include a sample receiving zone 108, a reagent zone 112, a flow channel 116, which can contain at least one detection zone (not shown), and an absorbing or wicking zone 120, respectively. In this specific device version, the fluid flow path is defined by a folded configuration extending from the sample receiving zone 108 through a reagent zone 112 containing a deposited detection material, such as a detection conjugate or other suitable reagent. The fluid flow path further extends along the flow channel 116 of the device 100, the latter further extending to a wicking or receiving zone 120 that defines the opposite end of the folded lateral fluid flow path. According to this particular device configuration, two distinct folds are present, a first fold between the reagent zone 112 and a first or entry end of the flow channel 116 and a second fold between a second or exit end of the flow channel 116 and the wicking zone 120.
According to this particular design a plurality of projections, similar to those previously depicted in FIG. 1, extend upwardly from the top surface of the substrate 104 substantially defining the active zones defined within the bordering line of this device 100 wherein the projections are specifically designed dimensionally in terms of their height and diameters, as well as with relative interpillar spacings, so as to solely promote spontaneous lateral capillary flow along the defined fluid flow path between the sample addition zone 108 and the wicking zone 120. As discussed infra, this specific device design is referred to as an “open” system or device, meaning that side walls and a cover are not necessarily required to assist in the creation of capillary force and as described in the following: U.S. Patent Application Publication No. 2014/0141527 A1; WO 2003/103835, WO 2005/089082; WO 2005/118139; WO 2006/137785; and WO 2007/149042, previously incorporated by reference in their entireties. It will further be noted that a cover or lid can be optionally included; for example, a cover (not shown) can be added to the device as needed, the cover being spaced in relation to the projections so as not contribute to the lateral capillary flow of a sample liquid. It is has been determined, however, similar to that depicted in FIG. 2, that the addition of a hydrophilic foil or layer 130 directly onto at least a portion of the wicking zone 120 alone does contribute to the overall flow rate (process time) of an aspirated sample.
The operation of this lateral flow assay device 100 is similar to each of the prior versions described. A fluidic sample, such as whole blood, is applied to the device 100 at the sample receiving area 108 such as through a cover (not shown) having an aperture (not shown) and separation filter (not shown). Upon contact with the projections of the sample receiving area 108, the sample is caused to move under capillary force along the fluid flow path through the reagent zone 112 in which the sample dissolves the deposited detection material to produce a visually perceivable signal. The sample and dissolved detection material then advance along the folded flow channel 116 to the absorbing zone 120, as further drawn due to the influence of the hydrophilic foil cover 130. Once it has been determined that the absorbing zone 120 is filled with sample, the detection instrument (not shown) can be used to determine analyte results through scanning or other means along the flow channel 116, which includes at least one detection area.
According to certain aspects, the fluid flow path of the assay device can alternatively include a porous material, e.g., nitrocellulose, in lieu of projections and define at least a portion of the flow path capable of supporting capillary flow. Examples include those shown in U.S. Pat. Nos. 5,559,041, 5,714,389, 5,120,643, and 6,228,660, all of which have been incorporated herein by their entireties.
An exemplary design of yet another known lateral flow assay device 300 is depicted in FIG. 4. This assay device 300 is defined by a planar substrate 304, which is made from a non-porous material, such as a molded plastic. As in the prior described assay devices, a plurality of zones or areas are disposed a defined fluid flow path. More specifically, a sample receiving zone 308 receives sample from a liquid dispenser, such as a pipette or other suitable means (not shown). The sample (e.g., whole blood) is typically deposited onto the top of the sample addition zone 308 through a cover (not shown) having an aperture (not shown). The sample receiving zone 308 is capable of transporting the dispensed liquid sample from the point when the sample is deposited to a pair of parallel spaced reagent zones 312, 313 through an optional filter and adjacent reagent addition zone 315, preferably through capillary flow. The capillary flow inducing structure can include porous materials, such as nitrocellulose, or preferably through a plurality of projections, such as micro-pillars or microposts that can spontaneously induce capillary flow through the assay device 300, in the manner previously described and shown in FIG. 1 and the prior incorporated US Patent Application Publication No. 2014/0141527 A1; WO 2003/103835, WO 2005/089082; WO 2005/118139; WO 2006/137785; and WO 2007/149042. A separation filter (not shown) or filter material (not shown) can be also be placed within the sample addition zone 308 to filter particulates from the sample or to filter red blood cells from blood so that plasma can travel through the assay device 300 as a filtrate.
As noted and located between the sample addition zone 308 and a folded portion of the flow channel 317 are the pair of adjacent reagent zones 312, 313, which are aligned in parallel relation herein. For purposes of the lateral flow assay devices herein described, including the improved versions, the reagent zones 312, 313 can include reagent(s) integrated into this analytical element and are generally reagents useful in the reaction—binding partners such as antibodies or antigens for immunoassays, substrates for enzyme assays, probes for molecular diagnostic assays, or are auxiliary materials such as materials that stabilize the integrated reagents, materials that suppress interfering reactions, and the like. Generally, one of the reagents useful in the reaction bears a detectable signal as previously discussed herein. In some cases, the reagents may react with the analyte directly or through a cascade of reactions to form a detectable signal such as a colored or fluorescent molecule. In this device design, the reagent zones 312, 313 each include a quantity of deposited conjugate material. The term “conjugate” as used herein means any moiety bearing both a detection element and a binding partner.
For purposes of this description throughout, a “detection element” is an agent which is detectable with respect to its physical distribution and/or the intensity of the signal it delivers, such as but not limited to luminescent molecules (e.g., fluorescent agents, phosphorescent agents, chemiluminescent agents, bioluminescent agents and the like), colored molecules, molecules producing colors upon reaction, enzymes, radioisotopes, ligands exhibiting specific binding and the like. The detection element, also referred to as a label, is preferably chosen from chromophores, fluorophores, radioactive labels and enzymes. Suitable labels are available from commercial suppliers, providing a wide range of dyes for the labeling of antibodies, proteins and nucleic acids. There are, for example, fluorophores spanning practically the entire visible and infrared spectrum. Suitable fluorescent or phosphorescent labels include for instance, but are not limited to, fluoroceins, Cy3, Cy5 and the like. Suitable chemiluminescent labels include but are not limited to acridinium esters, or enzymes such as peroxidase or alkaline phosphatase coupled with suitable substrates such as luminol, dioxetane and the like.
Similarly, radioactive labels are commercially available, or detection elements can be synthesized so that they incorporate a radioactive label. Suitable radioactive labels include but are not limited to radioactive iodine and phosphorus; e.g., 125I and 32P.
Suitable enzymatic labels include, but are not limited to horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase and the like. Two labels are “distinguishable” when they can be individually detected and preferably quantified simultaneously, without significantly disturbing, interfering or quenching each other. Two or more labels may be used, for example, when multiple analytes or markers are being detected.
The binding partner is a material that can form a complex that can be used to determine the presence of or an amount of an analyte. For example, in a “sandwich” assay, the binding partner in the conjugate can form a complex including the analyte and the conjugate and that complex can further bind to another binding partner, also called a capture element, integrated into the detection zone. In a competitive immunoassay, the analyte will interfere with binding of the binding partner in the conjugate to another binding partner, also called a capture element, integrated into the detection zone. Example binding partners included in conjugates include antibodies, antigens, analyte or analyte-mimics, protein, etc.
Referring back to FIG. 4 and located in the fluid flow path, before or after the reagent zone 312 and before the detection zone is an optional reagent addition zone 315. The reagent addition zone 315 can allow the addition of a reagent externally from the device 300. For example, the reagent addition zone 315 may be used to add an interrupting reagent that can be used to wash the sample and other unbound components present in the fluid flow path into a wicking (or absorbing) zone 324. In a preferred embodiment, the reagent addition zone 315 is located immediately downstream from the reagent zones 312, 313.
Still referring to FIG. 4 and downstream from the reagent zones 312, 313 and the optional reagent addition area 315 and along the lateral folded fluid path defined by the flow channel 317 is at least one detection zone, which is in fluid communication with the reagent zones 312, 313. The detection zone(s) and/or the flow channel 317 may include a plurality of projections, such as those as described above and shown in FIG. 1. These projections are preferably integrally molded into the substrate 304 from an optically transparent plastic material such as Zeonor, such through an injection molding or embossing process. The width in the flow channel 317 in the detection zone according to this specific device design is typically on the order of about 0.5 mm-about 4 mm and preferably on the order of about 2 mm, although others can be prepared on the order of about 1 mm, provided sufficient signal for a suitable detection instrument, such as a fluorimeter, can be read even if the reagent plume does not cover the entire width of the detection zone.
For purposes of this description throughout, the at least one detection zone is disposed anywhere along the flow channel 317 where any detectable signal can be read, although preferably the detection zone is located at about the center of the axial length of the flow channel 317 In a preferred embodiment and attached to the projections in the at least one detection zone are capture elements. The capture elements can hold binding partners for the conjugate or complexes containing the conjugate, as described above depending on the assay (e.g., competitive, sandwich). For example, if the analyte is a specific protein, the conjugate may be an antibody that will specifically bind that protein to a detection element such as fluorescence probe. The capture element could then be another antibody that also specifically binds to that protein. In another example, if the marker or analyte is DNA, the capture molecule can be, but is not limited to, synthetic oligonucleotides, analogues, thereof, or specific antibodies. Other suitable capture elements include antibodies, antibody fragments, aptamers, and nucleic acid sequences, specific for the analyte to be detected. A non-limiting example of a suitable capture element is a molecule that bears avidin functionality that would bind to a conjugate containing a biotin functionality. Multiple detection zones can be used for assays that include one or more markers. In the event of multiple detection zones, the capture elements can include multiple capture elements, such as first and second capture elements. The conjugate can be pre-deposited on the assay device 300, such as by coating or by deposition in the reagent zones 312, 313. Similarly, the capture elements can be pre-deposited on the assay device 300 on the at least one detection zone. Preferably, both the detection and capture elements are pre-deposited on the assay device 300, or on the reaction zones 312, 313 and detection zone(s), respectively.
For purposes of background, a brief treatment of the general process of the known lateral flow assay device 300 will now be generally discussed. After a predetermined quantity of sample has been delivered to the sample addition zone 308, the sample will be caused to migrate laterally along the defined flow path into the parallel disposed pair of reagent zones 312, 313. The sample will continue to flow under capillary action according to this version of device and interact with the detection material impregnated within the projections of the reagent zones 312, 313. As the sample interacts, the detection material begins to dissolve in which a resultant detectable signal is contained within the fluid flow, which is subsequently carried into the adjacent reagent addition zone 315. Alternatively and in lieu of the reagent zones, 312, 313, the sample can be combined with the reagent having the detection material prior to adding to the sample addition zone 308. According to this version, the detection material includes the conjugate having both the detection element and binding partner, in which case the perceived signal is often referred to as a “conjugate plume” and produces a fluorescent signal.
Downstream from the detection zone 318 and along the folded fluid path 317 is the wicking zone 324 in fluid communication with the detection zone. As in the case of prior lateral flow assay devices, the wicking zone 324 is an area of the assay device 300 with the capacity of receiving liquid sample and any other material in the flow path, e.g. unbound reagents, wash fluids, etc. The wicking or absorbing zone 324 provides a capillary force to continue moving the liquid sample through and out the intermediate detection zones of the assay device 300. The wicking zone 324 and other zones of the herein described device 300 can include a porous material such as nitrocellulose, or alternatively is a non-porous structure defined by projections as described previously. Though not shown, a hydrophilic foil cover can also be adhered or otherwise attached onto the absorbing zone 324 or the wicking zone 314 can further include non-capillary fluid driving means, such as an evaporative heater or a pump. Further details of wicking zones as used in lateral flow assay devices according to the present invention are found in patent publications US 2005/0042766 and US 2006/0239859, both of which are incorporated herein by reference in their entireties.
In this device version, the entirety of the fluid flow path of the assay device 300 including the sample addition zone 308, the reaction zones 312, 313, and the wicking zone 324 is defined by projections substantially vertical in relation to the substrate 304, and having a height, diameter and reciprocal spacing capable of creating lateral capillary flow of the sample spontaneously along the fluid flow path.
The defined flow path of the lateral flow assay devices described herein, including the device 300, can include open or closed paths, grooves, and capillaries. Preferably, the flow path comprises a lateral flow path of adjacent projections, having a size, shape and mutual spacing such that capillary flow is sustained through the flow path. In one embodiment, the flow path is in a channel within the substrate 304 having a bottom surface and side walls. In this embodiment, the projections protrude from the bottom surface of the flow channel. The side walls may or may not contribute to the capillary action of the liquid. If the sidewalls do not contribute to the capillary action of the liquid, then a gap can be provided between the outermost projections and the sidewalls to keep the liquid contained in the flow path defined by the projections. Preferably, the reagent that is used in the reaction zones 312, 313 and the capture members or detection agent used in the detection zone is bound directly to the exterior surface of the projections used in the herein described assay device 300.
Tests (assays) are typically completed when the last of the conjugate material has moved into the wicking area 324 of the lateral flow assay device 300. At this stage, a detection instrument, such as a fluorimeter or similar device, is used to scan the at least one detection zone, the detection instrument being movable and aligned optically with the flow channel 317 along an axis 319. The detection instrument that can be used to perform the various methods and techniques described herein can assume a varied number of forms. For example and as described according to the present embodiment, the instrument can be a scanning apparatus that is capable of detecting fluorescence or fluorescent signals. Alternatively, an imaging apparatus and image analysis can also be used to determine, for example, the presence and position of at least one fluorescent fluid front of an assay device. According to yet another alternative version, infrared (IR) sensors could also be utilized to track the position of fluid position in the lateral flow assay device. For instance, an IR sensor could be used to sense the ˜1200 nanometer peak that is typically associated with water in the fluid sample to verify that sample had indeed touched off onto the substrate of the assay device. It should be readily apparent that other suitable approaches and apparatus capable of performing these techniques could be utilized herein.
For purposes of this embodiment, the detection instrument is incorporated within a portable (hand-held or bench top) testing apparatus that includes means for receiving at least one lateral flow assay device 300 and defining a scan path along the flow channel 317 and coincident with axis 319 relative to a light emitting element of the detection instrument, such as a laser diode and an optical system and filtering, having an optical axis and capable of providing quantitative measurement of fluorescent signal at predefined wavelengths as emitted from the assay fluorophores in the lateral flow assay device, and as discussed herein. Other devices or testing apparatus can also be used to retain a detection instrument for purposes of the herein described monitoring methods. For example, a mainframe clinical analyzer can be used to retain a plurality of lateral flow assay devices as described in U.S. Patent Application Publication No. 2013/0330713, the entire contents of which are herein incorporated by reference. In a clinical analyzer, at least one detection instrument such as a fluorimeter can be aligned with the flow channel 317 of the assay device 300 and provided, for example, in relation to an incubator assembly as a monitoring station in which results can be transmitted to a contained processor.
One exemplary flow monitoring methodology is now herein described. For purposes of this method and in the description that follows, a lateral flow assay device as previously described according to FIG. 4 is utilized, although other device configurations could be utilized, this embodiment intended to be exemplary of a more generic technique.
For purposes of this particular version, a pair of detection or reader apparatuses could be employed; namely, a first reader apparatus 331 that is linearly aligned with the linear section of the flow channel 317 containing at least one detection zone along axis 319 and a second reader apparatus 334 that is linearly aligned with the wicking zone 324 along a second axis 337. In each of the foregoing apparatus, a reader or detector such as fluorimeter can be translated along the respective axes 319, 337 relative to specific areas designated on the lateral flow assay device 300. Alternatively, a single reader apparatus (not shown) could be utilized, the reader apparatus having capability of translating longitudinally and laterally so as to selectively align with either detection axis 319 or 337.
Before sample is administered or otherwise dispensed, the lateral flow assay device 300 can first be assessed by performing a so-called “dry scan” or read using each of the first and second reader apparatus 331, 334 at specific areas of the lateral flow assay device 300. For purposes of this embodiment, readings are taken using the second reader apparatus 334 adjacent the entrance and exit of the wicking zone 324 at designated positions 351 and 355, respectively, and the first reader apparatus 331 takes a reading at the at least one detection zone. The purpose of the “dry scan” is to obtain a background signal level prior to dispensing sample and comparing the background signal to a known standard. Readings that exceed the background standard can be indicative of error conditions, such as device structural flaws or a premature leakage of reagent or previous use. In any event, determinations that are not within a suitable range of the background signal can be detected by either reader apparatus and cause the assay device 300 to be discarded.
Alternatively, or in addition to, immediately upon installation of the device into the testing apparatus and either before or after addition of sample to the device 300, readings are taken at the wicking zone, such as at the exit of the wicking zone 324, at a designated position 355. Readings that exceed the background standard can be indicative of error conditions, such as premature leakage of reagent or evidence of previous use. In any event, determinations that are not within a suitable range of the background signal can be detected and cause the assay device 300 to be discarded. More specific details relating to the foregoing assay device 300 are provided in U.S. Patent Application Publication No. 2014/0141527 A1, entitled: Quality Process Control Of A Lateral Flow Assay Device Based On Flow Monitoring, the entire contents of which is herein incorporated by reference.
There is a general and ongoing need in the field to improve the flow characteristics of lateral flow assay devices, such as those previously described. For example, the amount of sample which is applied to the devices of FIGS. 2-4 is typically of the order of about 10 to 200 microliters, wherein a considerable amount of this sample is wasted. It is a general goal in the field to minimize the quantity of sample required to apply in order to adequately perform a test, but without sacrificing accuracy or reliability of results obtained. In addition and in the case of the lateral flow assay device shown in FIG. 4, the dual reagent areas or zones merge via separate paths into the flow channel. There is a need for such devices and others to insure adequate mixing has occurred between an applied sample and reagent prior to detection. Still further, it has been determined that the configuration of projections that creates a suitable amount of capillary force for moving sample through the device along a defined fluid flow path additionally creates a preferred distribution pattern in regard to an applied detection material. It would be advantageous to utilize this configuration in order to preferably and repeatably retain the deposited detection material in a defined area and also to more uniformly dissolve the deposited material when contacted with moving sample. Still further and with a need to use reduced amounts of sample, the time required to produce an assay result can be insufficient for certain markers, meaning additional time may be required for the fluidic sample to completely fill the absorbing zone which can be important, for example, for purposes of conducting test measurements for an analyte of interest. As discussed herein, the use of a hydrophilic cover improves the wicking ability of sample through the assay device. The edge of this cover, however, induces effects that are contrary to performance of the assay device. To that end, features are further required to hinder these effects and thereby improve reliability.